Antibody-Conjugated Nanoparticles For Biomedical Applications

Hindawi Publishing Corporation
Journal of Nanomaterials
Volume 2009, Article ID 439389, 24 pages
doi:10.1155/2009/439389
Review Article
Antibody-Conjugated Nanoparticles for Biomedical Applications
Manuel Arruebo,1 Monica

Valladares,2 and Africa
Gonzalez-Fernandez2
1 Aragon
Nanoscience Institute (INA), University of Zaragoza, 50009 Zaragoza, Pedro Cerbuna 12, Spain
Area, Faculty of Biology, University of Vigo, Campus As Lagoas-Marcosende, 36310 Vigo, Pontevedra, Spain
2 Immunology
Correspondence should be addressed to Manuel Arruebo, arruebom@unizar.es

Received 19 June 2009; Accepted 2 September 2009
Recommended by Maryam Tabrizian
Nanoscience and Nanotechnology have found their way into the fields of Biotechnology and Medicine. Nanoparticles by themselves
oer specific physicochemical properties that they do not exhibit in bulk form, where materials show constant physical properties
regardless of size. Antibodies are nanosize biological products that are part of the specific immune system. In addition to their
own properties as pathogens or toxin neutralizers, as well as in the recruitment of immune elements (complement, improving
phagocytosis, cytotoxicity antibody dependent by natural killer cells, etc.), they could carry several elements (toxins, drugs,
fluorochroms, or even nanoparticles, etc.) and be used in several diagnostic procedures, or even in therapy to destroy a specific
target. The conjugation of antibodies to nanoparticles can generate a product that combines the properties of both. For example,
they can combine the small size of nanoparticles and their special thermal, imaging, drug carrier, or magnetic characteristics with
the abilities of antibodies, such as specific and selective recognition. The hybrid product will show versatility and specificity. In
this review, we analyse both antibodies and nanoparticles, focusing especially on the recent developments for antibody-conjugated
nanoparticles, oering the researcher an overview of the dierent applications and possibilities of these hybrid carriers.
Copyright 2009 Manuel Arruebo et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
1. Introduction
According to Lux Research, in 2006, governments, corporations, and venture capitalists worldwide spent $12.4 billion
on nanotechnology research and development up almost
30% from 2005. By 2014, Lux estimates $2.6 trillion or about
15% of the total global output in manufactured goods will
incorporate nanotechnology [1].
As many other industries, Nanoscience and Nanotechnology have also found their ways into the fields of Biotechnology and Medicine. The estimated global production
for engineered nanomaterials applied in Biotechnology will
reach by 2010 1 Tn/year, and by 2020 that value will increase
to 10 Tn/year [2]. Nanotechnology is already present in many
commercialized medical products. However, many of them
are not available directly to the consumer, instead, they are
used for research purposes [1].
Nanoparticles by themselves oer specific physicochemical properties that they do not exhibit in bulk form where
the materials show constant physical properties regardless of
their sizes. Those properties make nanoparticles applicable in
many biomedical applications. Compared to microparticles,
nanoparticles show larger surface area to volume ratio, which

implies that they may render higher binding eciencies.
Also, larger particles may block specific binding regions
on cells hindering cell receptors. However, some physical
properties are enhanced by using microparticles compared to
nanoparticles. For instance, if they are magnetic, larger particles are preferable due to their faster response to a magnetic
gradient. However, for any in vivo biomedical application
nonbiodegradable microparticles will be excluded due to
their potential accumulation in the spleen and the kidneys
due to the size exclusion capability of those organs.
The conjugation of dierent moieties to the nanoparticles widens their application fields and provides them with
new or enhanced properties. A range of biomoieties can
be conjugated to the nanoparticles including low molecular
weight ligands (folic acid, thiamine, dimercaptosuccinic
acid), peptides (RGD, LHRD, antigenic peptides, internalization peptides), proteins (BSA, transferrin, antibodies,
lectins, cytokines, fibrinogen, thrombin), polysaccharides
(hyaluronic acid, chitosan, dextran, oligosaccharides, heparin), polyunsaturated fatty acids (palmitic acid, phospholipids), DNA, plasmids, siRNA, and so forth.
2
Most of the mentioned moieties show proven selectivity
and clear advantages such as the ability of crossing biological
membranes. For instance, certain regions of the TAT-peptide
known as protein transduction domains are able to pass
biological membranes by mechanisms that are independent
of transporters and receptor-mediated endocytosis. The
susceptibility of the peptides to be denatured by proteases
and their short vascular circulation lifetimes are their main
drawbacks. Synthetic molecules of DNA or RNA known as
aptamers are also able to recognize specific proteins. The
main advantage of using aptamers compared to antibodies
is their in vitro selection process without using animals, in
addition to their small size, lack of immunogenicity, and
ease of isolation. The main disadvantages of using aptamers
compared to antibodies are the high cost to generate them
especially in large quantities and their susceptibility to
enzymatic degradation.
The conjugation of nanoparticles with antibodies combines the properties of the nanoparticles themselves with the
specific and selective recognition ability of the antibodies to
antigens. Also, the improvement in the cellular uptake as well
as the major intracellular stability may be two of the major
advantages of using antibody conjugated nanoparticles.
In this review we analyze the developments reached
by using antibody-conjugated nanoparticles, oering to the
reader a general scope of the dierent applications and
possibilities of those hybrid carriers. In addition, we describe
briefly the conjugation possibilities for those interested in
the specific chemistry involved. Nanoparticles conjugated to
peptides and proteins other than antibodies have not been
considered in this review. Conjugated dendrimers, dendritic
polymers, quantum dots, carbon nanotubes, and micelles
have been excluded in this review because each one is
deserving its own.
2. Antibodies: Structure and Function

Immunoglobulins or antibodies are a group of glycoproteins
that constitute one of the most important specific defence
mechanisms in vertebrate animals. All of them have a very
similar structure in a Y form (Figure 1) of bifunctional
molecules with two identical domains for antigen recognition (Fab fragment), and two identical domains with eector
functions (Fc fragment). The antigen-binding region is
highly specific, and varies among the antibodies. Thousands
of millions of dierent antibodies can be generated, each one
with a distinct specificity.
Antibodies have two identical light chains of 24-25 kD
(either, or, ) and two heavy chains, also identical, of 55
70 kD (, , , , or ) bound by disulphur bridges [3
6]. The type of immunoglobulin generated depends on the
type of heavy chain and, therefore, in vertebrate animals
five classes or isotypes of immunoglobulins (IgG, IgE, IgD,
IgA, and IgM) can be distinguished, each one with a distinct
functionality. In addition to the disulphur bridges between
the chains, there are intracatenary disulphur bridges that
provide stability to the molecule [7]. The light chain (L)
is formed by two domains of around 100 residues, known
as the variable domain (VL ), at its aminoterminal end, and
VH
CH 1
CDRs
VL
CL
Constant
region
Pepsine
Fragment F(ab)2
CH 2
CH 3
Papain
Fragments Fab
Fragment Fc
Figure 1: Structure of an Ig molecule carrying two heavy and two

light chains linked by disulphur bridges. The products resulting
from the action of enzymes (papain and pepsin) are shown.
the constant domain (CL ). The heavy chain (H) contains a

variable domain (VH ) and three or four constant domains
(CH 1, CH 2, CH 3, CH 4), depending on the isotype [8]. The
variable regions are the zones of the molecule involved in
antigen binding, where the most divergent hypervariable
regions, or complementary determining regions (CDR), are
three in the VL domain and three in the VH domain [9,
10]. These regions are separated by structural regions called
framework (FRW), which have highly conserved sequences.
Immunoglobulins (Igs) are found as membrane receptors in B lymphocytes, which are a subpopulation of leukocytes. After activation mediated by antigen (virus, bacteria,
parasite), and in collaboration with other immune cells (such
as T lymphocytes), the B lymphocytes become plasma cells
secreting antibodies. The antibodies can then be released
from the cell and have dierent eector functions.
Among their most important functions are the following:
(1) neutralization and blocking of pathogens and toxins;
(2) activation of complement: the IgM and IgG activate
a proteolytic cascade, which leads eventually to the
opening of pores in the membrane of the pathogens
or target cells;
(3) opsonization and Phagocytosis: the IgG are the main
type of antibodies capable of facilitating phagocytosis
of pathogens, by binding to receptors of macrophages
and dendritic cells;
(4) antibody-dependent cellular cytotoxicity (ADCC),
natural Killer (NK) cells, and macrophages lyse cells
in the presence of antibodies directed at target cells,
to achieve this, the Fc portion of the antibody binds
to the target cells, which secrete granular proteins that
mediate in cytolytic functions;
(5) responses to parasites and allergic responses: the
IgE is involved in allergic responses through the
activation of mastocytes, which release their charged
granules (of histamine, prostaglandins, etc.). It also
participates in the elimination of parasites, with the
help of eosinophils;
(6) Mucosal protection: the IgA undertakes mucosal
protection (respiratory, digestive) and is present in
secretions, such as breast milk.
3. Monoclonal Antibodies
In 1975, Cesar Milstein and George Kohler immortalized
antibody-producing B cells by their fusion with tumoral cells
to obtain hybrid cells or hybridomas (Figure 2).
These cells are capable of producing a single type of
antibody in large quantities; as they derive from a single
cell that divides into identical cells, the antibody that they
generate is known as a monoclonal antibody. The two
researchers were awarded the Nobel Prize for Medicine or
Physiology in 1984, for making possible the use of antibodies
directed against specific targets in large numbers, which has
been a revolution in many fields of knowledge, especially
biomedicine.
The production of monoclonal antibodies starts with
the repeated immunization of laboratory animals (generally
mice or rats), which are injected at intervals with the antigen,
against which it is intended to develop the corresponding
antibody. After several immunizations, when the B lymphocytes begin to proliferate in response to the antigen, the
activated lymphoid organ is removed, generally the spleen,
which has become a primary source of antigen-specific B
lymphocytes. Subsequently, the lymphocytes are fused with
mouse B tumoral cells (myelomas), which are capable of
proliferate indefinitely when they are cultivated in vitro
under specific conditions. However, they lack some of the
enzymes required for nucleotide synthesis and, also, are
unable to secrete their own endogenous Ig. The result is
a hybridoma, a cell that combines the capacity of the B
lymphocyte to produce a specific antibody with the capacity
of the myeloma cell to reproduce indefinitely. Hybridomas
can be frozen and thawed, allowing them to be kept for
an indefinite time. This means, among other attributes,
the availability of an inexhaustible source of specific and
homogenous antibodies.
Above all, monoclonal antibodies (mAbs) have meant
a revolution in the field of clinical diagnosis, for the
purification of products (coagulation factors, interferon), for
the design of new technologies, and also they have been
actively introduced into the treatment of various diseases.
In diagnosis and research, they have helped to increase
knowledge of a large variety of molecules, to define the
stages of leukaemia, to identify tumours and transcription
factors, to define species, to quantify hormones, to study
blood groups, to characterize infectious agents, besides an
enormous number of targets that are now known thanks to
monoclonal antibodies. In order to standardize the names
given to the antibodies produced against the cell membrane,
especially that of leukocytes, an agreement has been reached
to group under the same cluster or number all those
antibodies that recognize the same membrane molecule.
The cluster dierentiation (CD) concept has emerged as
a consequence of this agreement, in which, to date, more
than 300 dierent molecules have been classified. These are
revised and updated every four years at an international CD
3
Myeloma
culture
Mouse
splenocytes
Hybridoma Hydridoma
culture
Monoclonal
antibody
Figure 2: Schematic description of the synthesis of monoclonal

antibodies.
workshop. As examples, a helper T lymphocyte is known

as CD3+ CD4+ for presenting those membrane molecules,
which are recognized by specific antibodies; a cytotoxic T
lymphocyte is CD3+ CD8+, and a B lymphocyte is CD19+
CD20+. These and many other markers have improved the
study of cell subpopulations, have allowed better definition
of dierentiation states, and have helped in the identification
of leukaemia, lymphomas, breast tumours, and so forth.
In addition to the antibodies directed against molecules
of the cell membrane, there is a long list of antibodies
that recognize transcription factors, pathogenic agents,
hormones which are now essential in the laboratories of
Immunology, Microbiology, Physiology, Virology, Pathology,
Cell Biology, and many others, and are mainly used in
diagnostic techniques, such as ELISA, immunofluorescence,
inmunohistochemistry, Western blot, immunochromatography or lateral flow, nephelometry, agglutination and
precipitation methods, as some examples.
4. Monoclonal Antibodies in Therapy

The enormous therapeutic potential of mAbs was initially
undermined by certain technical diculties, which explain
the long gap from obtaining mAbs in suciently large
quantities in 1975 to the beginning of their clinical application. This started in 1986, with the use of the anti-CD3
antibody for avoiding rejection in heart transplantation,
and it was not until 1997 that mAbs entered in the
field of onco-haematology, when the US Food and Drug
Administration (FDA) approved Rituximab (a humanized
anti-CD20 monoclonal antibody) as the first antibody for
antitumoral therapy (http://www.fda.gov/).
In antitumoral therapy, when compared to conventional
chemotherapeutic agents, mAbs are high molecular weight
proteins with slow distribution kinetics and a limited tissuepenetrating ability. However, their enormous advantage is
that they are specific, thereby minimizing secondary eects,
as they would be directed at their tumoral targets (antigens)
without aecting or with only minimum eect on healthy
tissues. The capacity of monoclonal antibodies to penetrate
tumours or to access inflammation sites is low [11, 12]. In
particular, in antitumoral therapy, antigen expression and
blood irrigation are the factors limiting the eectiveness of
the mAbs. Therefore, for optimal eectiveness, the target
antigen should be tumour-specific, intensely expressed, and
its expression should not be susceptible to being reduced.
The mAbs requirements for achieving a maximum response
would be: a long half-life, the power to penetrate, not to
induce an immune response, and a maximum cytotoxic
eectiveness [13].
4
4.1. First-Generation Monoclonal Antibodies. The first clinical trials were carried out with mouse or rat mAbs
against receptors on the surface of human cells, such as
T lymphocytes (anti-CD3), B lymphocytes (anti-CD20),
and so forth. However, given that the sequences of these
antibodies are murine, their ecacy was aected by the
production of Human Anti-Mouse or Anti-Rat Antibodies,
HAMA or HARA, respectively, in patients who received
this therapy. The generation of this immune response is
the main impediment to therapeutic success with murine
mAbs, as they can cause important allergic reactions in
continuous treatments, or a reduction in their therapeutic
ecacy. Further limitations are their short half-life and the
diculty of the interaction between murine antibodies and
human cells of the immune system.
In an attempt to partially resolve this problem, antibody
fragments were used rather than an intact antibody. Specifically, the use of Fabs (fragment antigen binding) should
be mentioned, which include the variable regions of both
the heavy chains (Hv) and the light chains (Lv), as well
as the first constant domain of both chains (CH and CL).
These fragments do not include the Fc region, and this
means the immunogenicity that can be produced is lower
than if an intact antibody were used. The Fab fragments,
which provide the specificity of antibody, could be used for
sterically blocking or impeding the function of the target
antigen, or for modulating cellular function (by the binding
of a target antigen capable of translating intracellular signal).
However, the lack of the Fc fragment in the antibody impedes
the various functions specific to this region (to facilitate
phagocytosis, complement activation, antibody-dependent
cellular cytotoxicity), therefore, depending on the desired
aim, the therapy could become useless.
4.2. Second-Generation Monoclonal Antibodies. The HAMA
or HARA responses could be avoided if the monoclonal
antibodies were completely human. However, dierent technical diculties did not allow obtaining completely human
monoclonal antibodies using the traditional cell fusion
technique for producing hybridomas. In the mid 1980s,
molecular biology techniques provided a very important
tool, which allowed the production of what have been called
second-generation monoclonal antibodies or recombinant antibodies [14]. These antibodies are produced by
immortalization of the genes codifying the monoclonal
antibody molecule, instead of immortalizing the monoclonal
antibody-producing cell.
Two characteristics of these proteins have facilitated the
conceptual modelling, design and engineering of Ig-based
artificial molecules. At the genetic level, the structural genes
that codify the Igs are suitable for working in Molecular
Biology. These genes are organized as discrete exons, which
correspond to complete domains in the protein, separated
by intronic regions. Therefore, it is simple to manipulate
the intronic sequences by adding changes that allow the
introduction into the gene of new sequences in specific and
previously selected locations, without any risk of altering
the coding sequence present in the exons. Similarly, at
protein level, the structural-functional organization of the
Igs is structured into discrete modules with variable (V) and
constant domains (C) in both the heavy (H) and light (L)
chains, which has made the achievement of these objectives
easier.
Hence, it is possible to clone two regions, VH and VL ,
responsible for a required specificity, and assemble them as a
functional antibody in any class or subclass of human Ig [15].
A considerable number of expression systems are currently
available for this purpose [16]. Several eukaryotic cell lines
adequately support all the necessary and associated processes
for the expression, synthesis, assembly and post-translation
modifications of the recombinant antibodies [17].
In prokaryote organisms, detailed knowledge of the
genome, architecture and life cycle of filamentous bacteriophages [18, 19] and of the lambda phage [20, 21] has
also allowed the ecient expression of the DNA codifying
for antibodies in these phage and subsequent infection of
bacteria [22, 23]. Thanks to these phage, it has been possible
to generate very large antibody repertoires (libraries) [24, 25]
and, therefore, it has become possible to artificially imitate
the selection strategy employed by the humoral immune
system. Currently, this technology oers one of the most
powerful ways for creating artificial antibodies with reduced
immunogenicity and for the creation of antibody repertoires
in combinatorial gene banks with a similar size to the natural
repertoires.
4.2.1. Complete Antibody Molecules. These antibodies are
very similar to natural ones, with the two structural elements
present, thereby permitting the functionality of the molecule
with Fab and Fc portions. Primatized, humanized and
chimeric antibodies (Figure 3) are included under this heading. A chimeric antibody is a combination of sequences from
dierent species; the most common ones are those with a full
human sequence except for the variable regions, which are
of murine origin. In humanized antibodies, only some small
regions of the variable domains (called hypervariable regions
or CDRs) belong to the original species, and the remainder
have human sequences. Finally, primatized antibodies are
composed of variable regions of the primate and constant
regions of human origin [26].
This variety of antibodies requires a new nomenclature for identifying their origin. Hence, the sux-momab
indicates murine origin; the sux-ximab implies that it is
a chimeric monoclonal antibody; and -zumab designates
humanized monoclonal antibodies. In many cases, a further
complication is the small case letters that precede them, such
as h or hu (humanized), r (recombinant), or c (chimeric).
Chimeric Antibodies. A chimeric antibody is an artificial
molecule where the constant portions of the heavy and
light chains are from a human Ig, and the variable regions,
VH and VL , are obtained from a mouse or rat monoclonal
antibody. The aim of this construction is to reduce the
immunogenecity of the mouse or rat antibodies, without
aecting the specificity of the original antibody. The most
immunogenic regions of the antibody molecule are associated to the constant portion of the molecule, the Fc portion
[27]. These regions are replaced by equivalents of human
Genetic
construction
5
VH VL
Linker
(Gly4 Ser)3
VH VL VH A VL B VH B VL A VH VL VH VL CH 3
3 5
3 5
3 5
3 5
3
Linker
(Gly4 Ser)
AC
AC
AC
Protein
AC
AC
AC
AC
ScFv
Diabody
AC
Bispecific
antibody
AC
configuration is altered, but humanized antibodies have the

advantages of being less immunogenic, more eective and
have a longer half-life (between 3 and 24 days) than chimeric
antibodies (between 4 and 15 days) [11, 13].
AC
Triabody Minibody
Figure 3: Genetic construction of dierent antibody fragments.
origin so that the resulting molecule is less strange for

humans and, therefore, makes its use in vivo easier for
prophylaxis and treatment of human diseases. The term
chimeric, derived from chimera, the mythical monster born
from the union of Typhon and Echidna with the head of a
lion, the body of a goat and the tail of a serpent, was chosen
for this type of antibody, as they originate from two dierent
species (humans and mice).
Chimerization developed by Morrison et al. [28] and
Boullianne et al. [29] in the mid-1980s was based on the
cloning of murine VH and VL genes and their insertion
in eukaryotic expression vectors. Previously, the coding
genes of the constant portion of the human heavy and
light chains had been incorporated into these eukaryotic
expression vectors, with approximately 34% of the sequence
still being murine and the rest of human origin [27, 30].
These vectors are finally transfected in a stable form into
a selected cell line. The reverse transcriptase-polymerase
chain reaction (RT-PCR) technique is used for cloning the
VH and VL genes. Messenger or total RNA obtained from
a secretor hybridoma of the required specificity is used
as mould, and complementary oligonucleotides at the 3
and 5 ends/termini of each gene as initiators. It has been
demonstrated that the protein product generated by these
constructions is capable of recognizing and binding to the
antigen, and eciently mediating in eector functions, such
as complement activation and interaction with Fc receptors,
while showing a reduced immunogenicity [31]. Currently,
nearly half of the antibodies approved for therapy by the FDA
are of the chimeric type [32].
Humanized Antibodies. Unfortunately, some chimeric
molecules are still able to induce a humoral immune
response when administered to humans. Important Human
Anti-Chimeric Antibody (HACA) responses have been
found in 40% of the products used in humans [32]. Eorts to
reduce the immunogenicity of antibodies led to the creation,
between 1988 and 1991, of humanized antibodies. This
technology includes a variety of procedures, such as CDR
grafting, Ab reshaping, and Ab resurfacing/veneering [33
35], based on the substitution of the residues of the anchor
domains or Framework of the murine variable region by
human sequences, until only the three hypervariable regions
(510% of the sequence) are of murine origin and all the
rest of human origin [36]. The problem is that manipulation
of the murine hypervariable regions and their insertion into
the human anchor domains, generally entails a reduction
in antibody anity [33, 37], as its three-dimensional
Primatized Antibodies. Primatized antibodies are the result

of a chimeric structure of human and primate origin, as
a nearly exact copy of the human antibody with reduced
immunogenicity and the possibility of using repeated
doses and a long-term therapy [26]. The variable regions
come from Macaca fascicularis (Macaca irus, Cynomolgus
macaque), which are nearly indistinguishable from the
human equivalents, and the constant regions are of human
origin. Keliximab and Clenoliximab are examples of this
type of antibody, both in a trial phase [38, 39], and are
directed against a molecule present in helper T lymphocytes.
Lumiliximab is another antibody of this type, an anti-CD23
antibody that inhibits the production of IgE and is a potent
therapy in allergic processes [40].
4.2.2. Recombinant Molecules Derived from Antibodies.
Molecular biology has also been utilized (Figure 3) to
develop a more heterogeneous group of new proteins, all
based on the structure of antibodies, either as autonomous
recombinant entities (Fab type fragments, Fv of the simple
chain form of scFv, diabodies, triabodies, bispecifics, minibodies, phage antibodies), or as fusion proteins or conjugated
antibodies, in which the Fc or the Fab portion is combined
with new properties that have a toxin, an enzyme, a cellular
receptor, a cytokine, and so forth.
Antibody Fragments. Antibody fragments oer some advantages over intact antibodies in therapy, especially against
solid tumours. The speed of penetration by a fragment versus
an intact molecule is the most remarkable advantage. In
1988, Jain [11] established that an intact molecule of IgG
took 54 hours to penetrate 1mm into a solid tumour, while a
Fab fragment managed the same distance in 16 hours.
Although the expression of chimeric and humanized
antibodies has been carried out in eukaryotic cells, bacteria
(Escherichia coli) have been used more frequently for the
production of recombinant antibody fragments [41]. The
expression of antibody fragments in the periplasm of bacteria
follows an assembly pathway similar to that of the production
of conventional antibodies in the endoplasmic reticulum of
a B cell [41]. This allows producing molecules in a small
volume and in a subcellular compartment that is relatively
free from active proteolytic enzymes.
Single Chain Fv (scFv). Two studies published in 1988
showed the production in E. coli of a recombinant version
of the Fv fragment in which the VH and VL domains
were bound physically by a small and flexible peptide
linker [42, 43]. This linker, of around 15 amino acids, has
the necessary length and flexibility to permit an adequate
spatial orientation of the VH and VL , domains, generating
a functional Fv of around 25 kD. Such constructions were
called single chain Fv (scFv), which are more stable than
conventional Fv and conserve the capacity of recognizing and
6
binding to the antigen. Their small size means that their renal
clearance is rapid; therefore, they are potentially useful as
radioactively-marked molecules [44, 45].
The establishment of antibody gene banks has allowed
the generation of antibody repertoires of such size and
diversity that it is possible to presume that all the natural
repertoire can be found within them. The use of the lambda
bacteriophage and, subsequently, of filamentous bacteriophages as vectors has been the key in the construction of
these enormous versatile gene banks.
Diabodies, Bispecific Antibodies, and Triabodies. The Fab
and svFv fragments can have a limited use for certain
applications in which a high anity is required, as they
are molecules that have only one antigen combination site
(univalents). Multivalent versions of these fragments should
show a functional anity or higher avidity. In addition,
recombinant antibodies that combine two distinct specificities are particularly attractive for certain applications,
such as immunodiagnostics and immunotherapy, and several
strategies have been employed for producing dimers or
polymers of recombinant antibodies. Although some use the
chemical crossover of two individual fragments [46, 47],
previously prepared and separately purified, but with the
disadvantage of low yield and problems in the purification
of the required fragment, other more eective strategies
have recently been developed, based on recombinant DNA
techniques. One of these strategies is based on the scFv
construction in which the linker is smaller than the original,
which makes impossible the formation of a functional Fv
between the VH and VL domains in the same molecule. This
leads to the interaction of two molecules of the scFv, forming
a dimer, which has two antigen-combination sites. If the
VH and VL domains have the same specificity, the product
obtained is a bivalent homodimer known as a Diabody
[48]. Using the same technique, it is possible to produce
recombinant molecules with two dierent specificities, called
Bispecific Antibodies [49]. If the polypeptide that serves as
the linker between the VH and VL domains is completely
eliminated and both regions are expressed as a continuous
polypeptide, the result is the formation of a functional trimer
with the ability to bind to three antigen determinants. These
fragments have been called Triabodies [50, 51].
Minibodies. In 1994, Tramontano et al. [52] described the
design of an artificial polypeptide with a structure based
on the IgV domain, which was called a Minibody. This
polypeptide has a length of 61 amino acid residues and
adopts a conformation that has an antigen-binding ability,
and appears to show excellent pharmacokinetic properties in
tumours [53, 54].
Camelid Antibodies. The presence of antibody molecules
composed of heavy chain dimers devoid of the light chain
was discovered in camel blood [55, 56]. This motivated a
research group to create antibodies with a single VH domain.
To simulate the structure of the camel antibodies, mutations
were introduced into a pre-selected human VH domain, and
those changes were sucient to prevent the interaction of
the mutated human VH with the homologous human VL .
Subsequently, the sequence of the third hypervariable region
of the VH domain was randomized and the repertoire
obtained was expressed as a gene bank of recombinant
filamentous phage in E. coli. It was, thereby, possible to
obtain soluble VH domains capable of high-anity antigen
binding [57].
Fusion Proteins or Immunoconjugates. Recombinant DNA
technology has made possible the creation, design and
production of molecules called fusion proteins, in which
dierent functional and/or structural motifs are combined,
derived from two or more natural proteins. There are an
important number of fusion proteins, where fragments of
dierent antibody molecules are combined with toxins,
cytokines, adhesion molecules, components of the extracellular matrix, hormones, growth factors, CD molecules,
cellular receptors, and lipids.
Aptamers. Both oligonucleotide (sequences of oligonucleotides) and peptide (variable peptide sequence inserted
into a constant scaold protein) aptamers are also being
tested for their potential therapeutic use [58]. They have the
advantages that they are smaller than antibodies and easier
to design.
5. Human Monoclonal Antibodies

The advantages of being able to use fully human monoclonal
antibodies can be summarized as follows.
(i) To reduce the possibility of an immune response: the
administration of human immunoglobulins will not
lead to the development of strong immune responses,
such as those seen against mouse Igs, although antiidiotypic responses could appear.
(ii) The repertoire of antigenic epitopes is recognized in
a distinct way among the dierent species.
(iii) Human immunoglobulins interact better with
human eector systems, Fc receptors, opsonization,
and so forth, than antibodies of murine origin.
(iv) There are dierences associated to the distinct patterns of glycosylation among dierent species, which
can aect the eectiveness of the antibody as a
therapeutic agent.
(v) The half-life of these antibodies is longer. Human
antibodies have a half-life of 1124 days.
5.1. Immortalization of Human B Lymphocytes. The production of human monoclonal antibodies with the classic
hybridoma technique, using human myelomas or hybrid
fusions between human B-cells and mouse myeloma cells
(heteromyelomas), encountered numerous problems, above
all the instability of the hybridomas and the low production
of antibodies. This made the search for new methods to
obtain totally human antibodies imperative.
The first eorts were made by Steinitz in 1997, by the
tumoral transformation of human B lymphocytes using the
Epstein-Barr virus (EBV), a human oncogenic herpes virus
responsible for infectious mononucleosis and Burkitts lymphoma. In vitro studies have shown that infection with this
virus induces the activation and growth of B lymphocytes,
with antibody secretion, although in small quantities.
5.2. Transfection of Mammalian Cells. Another possibility
is the production of completely human monoclonal antibodies, by cloning both the heavy and light chains, and
their subsequent transfection to mammalian cells [59].
The perfection of the expression vectors, the identification
within the genome of appropriate integration sites and
the capacity to accumulate biomass mean that the cell
culture systems produce 1-2 g/L of antibodies [60]. The
mouse myeloma NSO and SP2/0 cell lines are those most
commonly used [61], although dierences have been found
in the glycosylation of the antibodies produced. Hence, for
example, these cells can add terminal galactoses with an
1,3 bond that is very immunogenic. That is why CHO and
monkey cells are currently used, as they lose the enzyme
responsible for the 1,3 galactosyl bond [62].
5.3. Use of SCID/Trimera Mice. Another technique is based
on the use of immunodeficient mice, such as Severe Combined Inmunodeficiency (SCID) mice, which have a severe
combined immunodeficiency due to an autosomal recessive
alteration of the enzyme responsible for the rearrangements
of the antigen receptors of the B and T lymphocytes. Various
research groups [6365] showed that fetal haematopoietic
tissues and mononuclear cells of human peripheral blood
settled in this type of mouse.
Another approach is the development of Trimera mice,
where the mice are submitted to total body irradiation
followed by the transplantation of bone marrow from SCID
mice for, subsequently, receiving human haematopoietic
tissue. Both types of mice have been used for obtaining
human monoclonal antibodies by introducing antigenspecific human lymphocyte cells, subsequent immunization
of the mouse, and the production of hybridomas by the
conventional technique [66]. However, keeping these mice is
dicult and requires special equipment and specific sterile
conditions, which are out of reach for many laboratories,
as these mice tend to suer from agammaglobulinemia,
severe lymphocytopenia and frequent infections due to their
immunodeficiency. In contrast, they are used for studying
diseases or implanting tissues of other animal species, as the
functioning of the monocytes, granulocytes and NK cells is
normal.
5.4. Use of Chicken Eggs. The production of human monoclonal antibodies using chicken eggs has recently been
described. The technique is based on the insertion of the
coding genes of the human antibody into embryonic cells,
and then introducing these cells into chicken embryos
[67]. The antibodies produced have similar biological and
physical characteristics as conventional antibodies. In addition, these antibodies lose their mucosal residue, increasing
7
their antibody-dependent cytotoxicity versus other types of
antibodies.
5.5. Use of Plants. Scientists from Jeerson Medical College,
Philadelphia, USA and from the Cuban Centro para el Control
Estatal de la Calidad de los Medicamentos (CECMED) (State
Centre for Drug Quality Control) have successfully tested,
in laboratory experiments, monoclonal antibodies obtained
from transgenic tobacco plants, which recognize and destroy
breast cancer cells and those of colon and rectal cancer.
Tobacco plants have been genetically transformed with
the human antibody against the Lewis Y antigen, which
is found in tumoral cells, making the plants factories
for producing human antibodies. It has been shown that
recombinant antibodies are at least as ecient as those of
mammalian cell cultures obtained using conventional methods, with the advantages of having much lower production
costs, permitting eventual mass production, and of being
safer in regard to possible contamination.
Clinical trials of these antibodies will be carried out
during the next 510 years.
5.6. Use of Transgenic Mice for Human Immunoglobulins: The
XenoMouse. Given the problems mentioned in obtaining
completely human antibodies, another possibility proposed
by scientists was the development of transgenic mice with
sequences of producer genes of human immunoglobulins,
which, therefore, were able to generate human-specific
antibodies. In the 1990s, and thanks to the development
of techniques in Molecular Biology, microinjection, and
manipulation of embryonic cells, a race began among
various research groups to produce genetically modified mice
that could produce human antibodies. This type of mouse
is known as XenoMouse and has human immunoglobulin
transgenes containing a large part of the V gene repertoire
in the terminal line, which supports the development of a
large population of B lymphocytes and the formation of
a broad and diverse primary immune repertoire, secreting
human antibodies [68, 69]. The human genes are compatible
with the factors that mediate the rearrangement of the genes
and with the mouse enzymes that mediate class switching,
the introduction of mutations (somatic hypermutation) and
anity maturity [70].
The vectors that have been used by dierent authors
for the introduction of human Igs in mice have essentially
been miniloci, P1 vectors, and yeast artificial chromosomes
(YACs) and, more recently, entire or almost entire chromosomes. While miniloci can incorporate a few Kb of DNA, the
YAC vectors can include large segments of exogenous DNA
(up to 1-2 Mb) and can be modified in a rapid and eective
way using recombination processes. The fundamental advantages of using spheroblast fusion that contains YACs are that
they do not require DNA purification processes, and that the
methodology is similar to the standard processes of somatic
fusion of mouse splenocytes for obtaining monoclonal antibodies. Using xenomouse secreting human antibodies our
group has developed several human monoclonal antibodies
directed against human leucocyte cells [7175].
6. Therapeutic Uses of Monoclonal Antibodies

Currently, therapy with monoclonal antibodies is the largest
growth area in the pharmaceutical industry. The development of Genetic Engineering and Immunology in recent
years, with the subsequent production of all the types of
antibodies described, is opening the door to hope for many
therapies.
Numerous patients are already benefiting from the use of
monoclonal antibodies as therapies against various diseases,
and there are many more where the antibodies are still
in dierent phases of clinical trials, prior to approval for
commercial therapy.
Antitumoral therapy stands out among the treatments
for diseases where monoclonal antibodies are currently being
used. Some of the reasons that have driven the search for
new therapeutic weapons are: the high incidence in the
population of dierent types of cancer; the extremely severe
nature of many of these cancers without adequate treatment;
the resistance of the tumoral cells to many treatments;
the absence of eective therapies; and the extreme toxicity
of certain current treatments. Among this new armoury,
monoclonal antibodies can oer a specific weapon that can
be directed against the tumoral cell without aecting, or
with only minimum eect, upon healthy tissues and with less
adverse eects than other therapies.
Tumoral cells generally express antigens on the outer
space, specific to their lineage, signalling proteins, growth
receptors, and for B-cell proliferative syndromes, membrane
immunoglobulins. Many of these antigens are identical to
those expressed by precursor cells or non-tumoral adult cells.
Therefore, to be good candidates as therapeutic targets, they
have to be overexpressed in target cells or, in some cases, be
specific to them. Hence, the success of the treatment with
antibodies depends upon the ability of the cells to tolerate
collateral damage or to be reconstituted by the negative
precursor cells for the target antigen [76].
Monoclonal antibodies can be therapeutically useful
through a variety of mechanisms, from the simple blocking
of the antigen receptor in the eector cells, to cytotoxic action
on the cells that express the corresponding antigen, with
numerous intermediate options of cellular modulation.
7. Monoclonal Antobodies Approved for

Therapeutic Use by the FDA
The Food and Drug Administration (FDA) of the United
States has approved 26 monoclonal antibodies for clinical use, either therapeutic or diagnostic (Table 1), while
around 150 are in dierent phases of clinical trials
(http://www.fda.gov/). The situation is similar in the European Union, as since 1995 when the European Medicines
Agency (EMEA) was set up, the registration process for this
type of drug is a centralized European procedure, which
coordinates the eorts for evaluation and control of these
very particular medicines.
Most of the antibodies that have, so far, been approved
are targeted against dierent tumoral processes and are
humanized, chimeric and murine antibodies (Table 1). The
list of monoclonal antibodies that are currently in dierent
phases of clinical trials means that there will be an increase
of around 100 monoclonal antibodies in 2010.
8. Nanoparticles and Antibodies

The antibody-conjugated nanoparticles can be used principally in two biomedical applications: therapy and diagnosis.
In therapy, the development of targeted drug delivery
represents, together with tissue repair, the main applications
of antibody-conjugated nanoparticles. In diagnosis, the
applications can be divided into those using in vivo and
those using in vitro experimentation (Figure 4), and include
contrast agents for magnetic resonance imaging (MRI),
sensing, cell sorting, bioseparation, enzyme immobilization,
immunoassays, transfection (gene delivery), purification,
and so forth. The importance of all these applications can
be demonstrated by a list of all the companies involved
in the synthesis and applications of antibody conjugated
nanoparticles (Table 2); for example, cell sorting and bioseparations are the main applications where a significant
number of companies are currently commercializing their
products.
9. Therapeutic Applications of
Antibody-Conjugated Nanoparticles
Commercial antibodies are already on the market either
attached to drugs (Mylortag ) or to radioisotopes (i.e.,
ProstaScint ), used in the treatment of acute myeloid
leukaemia and prostate cancer, respectively. However, to
date, there are no commercial antibodies conjugated to
nanoparticles applied in therapy, although the combination
of nanoparticles and antibodies can oer versatility together
with specificity (Figure 5), and there is a huge potential
market.
The combination of antibodies and nanoparticles has
been developed at a stunning pace, as can be observed in
Figure 6, where the number of articles (as indexed by ISI Web
of Knowledge ) published increased almost exponentially
during the last years (2009 data were not yet definitive when
the search was carried out (June19, 2009).
Reviewing those articles we can conclude that targeted
drug and gene delivery together with magnetic or optical
hyperthermia are the main potential therapeutic applications
for antibody-conjugated nanoparticles. Each one is in a
dierent development phase. All these applications have in
common the selective delivery of a therapeutic carrier, such
as a drug, a gene or heat, to cause the death of malignant
cells, the expression of a specific protein or the activation of
others, without aecting the surrounding healthy tissues.
Specific targeting is commonly achieved using passive or
active strategies. Passive targeting employs the characteristic
microvasculature of tumours to tailor the nanoparticles to be
accumulated on these targets. The nanoparticles accumulate
due to their specific size and their extravasation within
the tumour, where the microvasculature is hyperpermeable
and leaky, a process also aided by the tumour-limited
lymphatic drainage. In combination, these factors lead to
Table 1: Monoclonal antibodies approved for human therapy, their applications and data of approval by FDA and/or EMEA.
Product
Muromomab
(Orthoclone OKT3)
Company
Ortho Biotech, Inc.
(Johnson & Johnson)
Applications
Abciximab (ReoPro)
Nofetumomab
(Verluma)
Imciromab-Pentetate
(Myoscint)
Centocor B.V.
Boehringer Ingelheim
Pharma KG
Centocor (Johnson &
Johson)
Chimeric 7E3 directed against platelet glycoprotein IIb/IIIa

Mouse Fab IgG2b directed against glycoprotein 40 kD
(expressed in several tumours). Conjugated to Tecnecio99
Arcitumomab
(CEA-Scan)
Mouse IgG2a anti-CD3. Transplantation Rejection
Approval
FDA 1986
EMEA 1987
FDA 1994
FDA 1996
Mouse Fab-Indio111 , directed against human heart miosin
FDA 1996
Immunomedics Inc.
Mouse Ig Fragment-Tecnecio99 . Directed against

carcinoembrionic antigen
FDA 1996
EMEA 1996,
Retired 2005
Cytogen Corp.
Mouse Ab-Indio111 . Detection of prostate tumour
FDA 1996
Homan-LA Roche
Inc.
Humanized IgG1 anti-IL-2. To avoid transplant rejection
Rituximab (Rituxan)
Genentech Inc.
Chimeric Ig antiCD20 for Non-Hodgkin lymphomas
Basiliximab
(Simulect)
Novartis
Phamaceutical Corp.
Chimeric anti-IL-2(CD25). To avoid renal transplant
Palivizaumab
(Sinagis)
Medimmune Inc.
Humanized IgG1 ; respiratory syncitial virus
Trastuzumab
(Herceptin )
Genentech Inc.
Humanized IgG anti-HER2; breast cancer
Remicade
(Infliximab)
Centocor (Johnson &

Johnson)
Chimeric anti TNF-. Rheumatoid arthritis, Crohns disease
Wyeth Averst
Humanized Ig anti CD33; acute myeloid leukaemia
Millennium/ Ilex
Partners LP
Humanized Ig anti CD52; chronic lymphocitic leukaemia
Abbott Laboratories
Human anti TNF-. Rheumatoid arthritis
Capromab Pendetide
ProstaScint
Daclizumab
(Zenapax)
Gemtuzumab
Ozogamicin
(Mylotarg)
Alemtuzumab
(Campath)
Adalimumab
(Humira)
Ibritumomab
Tiuxetan (Zevalin)
Omalizumab (Xolair)
Tositumomab
(Bexxar)
Efalizumab (Raptiva)
Cetuximab (Erbitux)
IDEC
Pharmaceuticals
Corp.
Genentech Inc./Roche
Mouse Ig-Itrio 90 anti CD20. Non-Hodgkin lymphoma

Humanized IgE. Severe asthma
FDA 1997
EMEA 1999
FDA 1997
EMEA 1998
FDA 1998
EMEA 1999
FDA 1998
EMEA 1999
FDA 1998
EMEA 2000
FDA 1998
EMEA 2002
FDA 2000
FDA 2001
EMEA 2001
FDA 2002
EMEA 2003
FDA 2002
EMEA 2004
FDA 2003
EMEA 2005
GlaxoSmithKline
Murine Ig-Iodo131 anti CD20. Non-Hodgkin lymphoma
FDA 2003
Genentech Inc./Roche
ImClone Systems/
Bristol-Nyers
Squibb/Merck KgaA
Humanized Ig anti-CD11a
FDA 2003
IgG1 directed against EGFR; colorectal tumour
FDA 2004
Bevacizumab
(Avastin)
Genentech Inc.
Humanized anti VEG; tumours
Natalizumab
(Tysabri)
Biogen Idec
Humanized anti CD49d. Multiple Sclerosis, Chrons disease
Panibizumab
(Lucentis)
Genentech Inc.
Humanized anti VEGF-A. Wet Macular Degeneration
Panimumab
(Vectibix)
Amgen
Human anti EGFR (epidermal growth factor receptor).

Metastatic colorectal carcinoma
FDA 2004
EMEA 2005
FDA 2006
EMEA 2006
FDA 2006
EMEA 2007
FDA 2006
10
Table 1: Continued.
Product
Eculizumab (Soliris)
Certolizumab Pegol
(Cimzia)
Golimumab
(Simponi)
Company
Alexion
Pharmaceuticals Inc.
UCB Pharma
Centocor (Johnson &
Johnson)
Applications
Humanized anti CD59. Paroxysmmal nocturnal
hemoglobinuria
Humanized Fab anti-TNF-. Morbus Chron, rheumatoid
arthritis
Human anti TNF-. Rheumatoid psoriatic arthritis,
ctiveAnkylosing spondylitis
Approval
FDA 2007
EMEA 2007
FDA 2008
FDA 2009
Table 2: Companies involved in the synthesis and applications of antibody conjugated micro and nanoparticles.
Company
Ademtech SA
Alnis Biosciences Inc.
Bangs Laboratories Inc.

Chemicell
Diagnostic Biosensors, LLC
Indicia Biotechnology S.A.
Invitrogen Corp.
LifeAssays
Magnamedics GmbH
Magnisense SAS
MagSense Life Sciences, Inc.
Merck & Co., Inc. (Estapor )
Micromod Partikeltechnologie GmbH
Miltenyi Biotec GmbH
Nanosphere, Inc.
Spherotech Inc.
Stemcell Technologies
Triton Biosystems Inc.
Applications
Cell sorting, Biomagnetic Separation
Under development:
antibody-conjugated magnetic
nanoparticles for diagnosis and treatment
Biomagnetic separation of cells,
organelles, proteins, immunoglobulins,
nucleic acids, and so forth.
Binding of secondary antibody
Immunoassays, biosensors
Biomagnetic separation
Immunoassay, cell separation, binding of
secondary antibody
Magnetic biosensors
Immunoassays, biosensors
Magnetic bioseparation or
biopurification of antigens, antibodies or
nucleic acids
Biomagnetic separation of cells, indirect
magnetic labelling, isolation of apoptotic
and dead cells
Optical biosensors
Biomagnetic and no-magnetic separation
Under development or in pre-clinical
development: targeted nanoparticles for
hyperthermia
the selective accumulation of nanoparticles of sizes generally

between 80 and 200 nm in tumour tissue, a phenomenon
known as enhanced permeation and retention (EPR). In
general, the smaller the nanoparticle, the longer is its
circulation time. Although vasculature organization may
dier depending on the tumour type, its growth rate and,
microenvironment, most solid tumours exhibit a vascular
pore cut-o size between 380 and 780 nm [77]. Particles that
are too small (<80 nm) may be cleared very quickly from the
tumour vasculature by drainage through the capillary pores.
Therefore, there is an optimum size for the nanoparticles
to be accumulated in the proximity of a tumour. Usually,
passive targeting is applied by injecting the nanoparticles
Website
http://www.ademtech.com/
http://www.alnis.com/
http://www.bangslabs.com/
http://www.chemicell.com/
http://www.diagnosticbiosensors.com/
http://www.indicia.fr/
http://www.invitrogen.com/
http://www.lifeassays.com/
http://www.magnamedics.com/
http://www.magnisense.com/
http://www.magsenselifesci.com/
http://www.estapor.com/
http://www.micromod.de/
http://www.miltenyibiotec.com/
http://www.nanosphere.us/
http://www.spherotech.com/
http://www.stemcell.com/
http://www.tritonbiosystems.com/
near the artery that feeds the tumour, and produces good
results in terms of reducing the tumour size.
Active targeting is based on the over- or exclusiveexpression of dierent epitopes or receptors in the tumoral
cells, and on specific physical characteristics. In addition to
all the moieties mentioned in the introduction, antibodies
represent one of the most interesting bioconjugates used to
achieve the active targeted delivery of a therapeutic carrier.
Moreover, active targeting is successful using the physical
properties of the nanoparticles (i.e., magnetism) [78]. The
combination of dierent active targeting strategies, both the
physical and those aided by specific recognition moieties, is
also possible.
11
Antibody-conjugated nanoprticles
Therapy
Drug/Gene
delivery
Diagnosis
Hyperthermia/ thermal
ablation/
phototherapy
In vivo
In vitro
MRI
Sensing
Cell sorting
Tissue repair
Bioseparation
Enzyme
immobilization
Radiotherapy
Immunoassays
Transfection
Purification
Figure 4: General scheme of the dierent applications for antibody conjugated nanoparticles.
What antibodies can oer to NPs and what NPs can oer to Abs
Antibodies
Nanoparticles
Nano-size
Specificity
Biological component
Carriers
Recruits components
of the immune system
Can activate
the immune
system
Can find specific target

Specific
Not specific
Several properties
Thermal
Magnetics
Imaging
Drug delivery-controlled
Many properties
Versatility
Figure 5: Advantages in the conjugation of nanoparticles to antibodies.
9.1. Targeted Drug Delivery. Antibody-conjugated drugloaded nanoparticles can selectively target malignant cells
and release large amounts of a drug in the cell cytoplasm,
minimizing undesired side eects. The ecacy of the delivery
depends on the ability of each antibody to reach its target
in adequate quantities, and on the limited amount of
nanoparticles trapped by the cell. Since the early studies in
1977 of antifolates linked to cytotoxic drugs (methotrexate)
demonstrated therapeutic action in mice [79], the idea of
conjugating a drug-loaded nanoparticle to an antibody for
the targeted recognition of malignant tissue has opened the
development of new targeted nanocarriers. The need to use
nanocapsules (with a hollow interior filled with the drug)
depends upon the amount of drug required to cause cellular
death. For example, on the cell line MCF-7 it has been
shown that for 50% cell killing 107 molecules of a drug
(daunomycin) have to be present per cell [80]. Given that
the number of antigens recognised by tumour-associated

antibodies on cell surfaces is reported to be in the range
105 107 per cell, it would be dicult to deliver a sucient
amount of even the most potent conventional cytotoxic
agent through specific antibody delivery systems, solely with
antibodies [81]. Moreover, there appears to be an upper
limit for antigen-antibody binding anity. Beyond this limit,
further improvements in anity have no beneficial eects
[82].
The following are some examples of antibody-conjugated
nanoparticles used in targeted drug delivery.
(i) Gupta and Torchilin (2007) described the synthesis
and ecacy of the monoclonal anticancer antibody
2C5 conjugated to commercially available PEGylated liposomes loaded with doxorubicin (a DNAinteracting drug widely used in chemotherapy), in
12
350
Scientific publications
300
250
200
150
100
50
0
1999
2001
2003
2005
Year
2006
2008
(a)
Biochemistry & molecular biology

Science & technology-other topics
Chemistry
Immunology
Pharamacology & pharamcy
Instruments & instrumentation
Engineering
Cell biology
Physics
(b)
Figure 6: (a) Number of papers related to antibodies and nanoparticles published in each year in the 19992009 period of time. Topic
= (antibodies and nanoparticles). Note: 2009 is uncompleted (search done on 19/06/2009). (b) Ranking of the scientific publications by
subject area.
an intracranial model in nude mice [83]. The treatment with the antibody-conjugated liposomes provided a significant therapeutic benefit over controls,
with a pronounced reduction in the tumour size.
(ii) Poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles (PLGA/MMT NPs) have been decorated
with human epidermal growth factor receptor-2
(HER2) antibody, Trastuzumab (Herceptin ), for
targeted breast cancer chemotherapy with paclitaxel
as a model anticancer drug [84]. According to the
authors, their in vitro studies revealed that the
therapeutic eects of the drug formulated in the
conjugated nanoparticles could be 12.7 times higher
than that of the bare nanoparticles, and 13.1 times
higher than Taxol . The same humanised IgG1
monoclonal antibody has been covalently attached to
human serum albumin-based nanoparticles, and the
resulting carrier showed specific targeting to HER2positive breast cancer cells [85].
(iii) Paclitaxel-loaded poly(lactide-co-glycolide) (PLGA)
nanoparticles coated with cationic SM5-1 singlechain antibody (scFv), containing a polylysine
(SMFv-polylys), were synthesized and eectively
tested in vitro. The purpose of tagging the antibody
with the cationic polypeptide polylysine is to achieve
electrostatic attraction with the negatively charged
nanoparticles [86]. When compared to nontargeted
paclitaxel-loaded PLGA nanoparticles, the antibodyconjugated nanoparticles showed enhanced in vitro
cytotoxicity against human hepatocellular carcinoma
cell lines (Ch-hep-3).
(iv) Two dierent monoclonal antibodies have been covalently coupled to the same poly-(malic acid)-based
nanoparticle, one of them, monoclonal anti-TfR, to

direct the conjugate across the blood-tumour barrier,
and the other, monoclonal 2C5, to target tumourcell surface-bound nucleosomes. The presence of
both antibodies on the same nanoparticle and their
biological activity were confirmed by ELISA. In vivo
experiments showed the significantly higher accumulation of the conjugated nanoparticles in human
glioma [87].
(v) Tyner et al. [88] have recently reported the surface
functionalization of inorganic nanoparticles (made
of magnesiumaluminium layered double hydroxides) with disuccinimidyl carbonate (DSC), which
were then loaded with a huA33 antibody and a blood
plasma protein (serum albumin). The biological in
vitro tests showed that LDH-DSC-huA33 nanobiohybrids had an activity against human A33 antigen 30
times higher than that of LDH-DSC-albumin.
9.2. Gene Delivery and Tissue Repair. Excluding rare exceptions, bare DNA does not internalize the cells nor does it
express a specific protein at a reasonable level. The carriers
that help the DNA to internalize into the cell can be
administered systemically or locally (by direct injection in
the tissue or by using DNA-loaded scaolds). Viral carriers,
cationic lipids and polymers, recombinant proteins, and
inorganic nanoparticles are the four kinds of carriers used.
Some of these vectors may be functionalized with antibodies
or Fabs for targeting cell delivery, while others, with short
peptide sequences, act as nuclear localisation signals.
Nanoparticles have been used as carriers of plasmid
DNA, oligonucleotides, siRNA, and so forth, to transfect
cells and use the cellular machinery to produce therapeutic
proteins, to activate a cellular response or to dierentiate
the cells into specific ones. The nanoparticle can behave as
a protective coating to the moiety. For instance, synthetic
siRNAs are a very promising new tool for therapeutic
intervention; however, it is necessary to improve their
cellular target specificity, their eective cellular delivery
(particular into primary cells), as well as their stability
[89].
Hayes et al. [90] described the use of an antibodylipopolymer (anti-HER2 scFv (F5)-PEG-DSPE) conjugated
to a cationic lipid nanoparticle with the DNA encapsulated
in its interior, which achieves a high degree of specific
transfection activity.
Several research groups, interested in the noted blood
instability of siRNA, have been working on their chemical
modification and on their encapsulation for protection
against degradation in transfection applications. One group,
Heidel et al. (2007) [91], described the use of transferrin
protein as a targeting ligand conjugated to cyclodextrinebased nanoparticles, which encapsulate siRNA for inhibiting
gene expression in nonhuman primates. Most of these gene
delivery carriers have applications in regenerative medicine.
Bare and antibody-conjugated gold nanoparticles that
absorb light in the near infrared have been shown to be
optically-absorbing nanoparticles for enhanced tissue repair
[92]. The heat adsorbing properties of gold nanocolloids are
used in this case to produce tissue welding.
9.3. Magnetic Hyperthermia, Thermal Ablation, and Photodynamic Therapy. Anticarcinoembryogenic antibodies of
human LoVo cancer cells have been attached to TiO2
nanoparticles [93]. The electrons in the valence band of TiO2
nanoparticles can be excited, under UV light irradiation, to
the conduction band, creating pairs of photo-induced electrons and holes. These photo-induced electrons and holes
present strong reduction and oxidation properties, which can
destroy malignant cells, due to the generation of reactive
oxygen species under ultraviolet irradiation. The antibodies
were labelled with FITC for improving optical detection
by using confocal laser microscopy [94]. Electroporation
(use of electric stimulation to deliver moieties through the
micropores on the cell membrane) was used to accelerate the
internalization of the conjugated nanoparticles into cancer
cells. In vitro results showed that 100% of human cancer cells
were photokilled within 90 min using the combination of
both techniques.
For the same application, polyacrylic acid-coated TiO2
nanoparticles were covalently coupled to antiestradiol mouse
antibodies with an amide bond, and used not only in the
recognition of the estradiol, but also in its destruction by
using the previously mentioned photocatalytic properties
of TiO2 . There are clear applications for these carriers in
environmental protection for water treatment technology,
as well as in medical and public sanitation waste-treatment
processes [95].
El Sayed et al. [96] showed selective laser photothermal
destruction of two oral squamous carcinoma cell lines (HSC
313 and HOC 3 clone 8), versus a benign epithelial cell line
(HaCaT), by using anti-EGFR antibody conjugated to pure
gold nanoparticles. Gold nanoparticles show heat-adsorbing
13
properties under near infrared radiation (NIR), whereas
tissue does not adsorb NIR light.
Staphyloccocus aureus bacteria were killed by lightadsorbing gold nanoparticles conjugated to anti-protein A
antibodies, using laser irradiation at 532 nm [97]. Protein
A was chosen because it interacts specifically with the Fc
fragment of the antibody. According to the authors, killing
eciency depends on the local overheating eects accompanied by bubble-formation phenomena. Direct irradiation of
bacteria with the laser did not damage the bacteria, because
of low absorption by natural endogenous cytochromes.
Gold-coated silica nanoparticles have been used in
phototherapy in the near infrared [98] showing a potential
application in tumours present on the external surface of
the body. The in vitro results showed an irreversible thermal
damage for human breast carcinoma cells incubated with the
nanoparticles after exposure to NIR light, whereas control
tissues appeared to be undamaged.
Pharmacokinetics, tumour uptake, and the therapeutic
eect of inductive heating (by externally applying alternating magnetic fields, AMF) of 111 In-chimeric L6 (ChL6)
monoclonal antibody-linked iron oxide nanoparticles were
studied in athymic mice bearing human breast cancer HBT
3477 xenografts. Tumour regression was observed at all
AMF levels after those carriers were delivered to the cancer
cells [99]. In magnetic hyperthermia, heat is produced by
increasing the alternating magnetic field amplitude and/or
field frequency, due to the high specific absorption rate of
magnetic nanoparticles.
The main disadvantage of magnetic hyperthermia is that
there is a limitation in the external field strength that can
be applied to create an adequate gradient to target the
magnetic nanoparticles in the desired area. The magnetic
gradient decreases rapidly with increased distance from the
external magnet. The same spatial limitation would occur for
phototherapy using NIR.
9.4. Encapsulation of Biomolecules for Bioanalytical, Therapeutic, and Environmental Purposes. Monoclonal antibodies
may have low physico-chemical stability and short in vivo
half-life. Their encapsulation or conjugation to nanoparticles may oer an alternative to avoid those problems.
Nanoparticles can also encapsulate a drug or protect it within
the pores of their structure, oering potential protection
from metabolism, enzymatic degradation, and filtration. In
addition, the use of nanoparticles to encapsulate the drug has
the clear advantages that the drug activity is not aected by
coupling reactions, and that they have an inherent high drugloading capacity.
The main application of encapsulated antibodies within
nanoparticles relies on their use as biosensors. Antibodies
are used for analytical purposes in immunoassays, selective
analyte enrichment (immunoanity extraction), and in
biosensors. In one study, anti-diclofenac antibodies were
encapsulated within porous silica nanoparticles prepared by
the sol-gel method [100]. The capsule provides the antibody
with protection without loss of biological activity. The same
authors show several antibodies encapsulated in dierent
matrices.
14
Special mention should be given to the use of nanoparticles to encapsulate antigens for vaccination purposes.
The capsule provides the antigen with enhanced solubility
and circulation time in the blood stream and, in some
cases, immunostimulation [101]. Nanoparticles may present
targeting ligands and sequestrate and release guest molecules
[102]. The ability to induce potent and specific HIV1 humoral and cellular immune responses in vivo has
been demonstrated for antigen-loaded poly(-glutamic acid)
nanoparticles [103]. Cationic nanoparticles have been used
as a nasal delivery system for two recombinant proteins
(HBsAg and -galactosidase), which require humoral, cellular and mucosal responses in vivo [104]. N-trymethyl
chitosan nanoparticles loaded with influenza subunit antigen
have been shown to be eective carriers for the enhanced
response of systemic and local immune response in mice
[105]. In this case, the local uptake is mainly based on
the mucoadhesive properties of the nanoparticles, due to
their electrostatic attraction to the negatively charged nasal
mucosa.
Simultaneous surface coating of two vaccine antigens
(HIV-1 p24 and gp120 proteins) on poly(lactic-co-glycolic
acid) or polylactic acid nanoparticles by adsorption was
used to prove their immunogenicity in mice as well as
their stability, assessed by a specific antibody binding assay
[106].
Single-chain Fv antibodies that specifically bind and
enter prostate cancer cells have been loaded within liposomes. The liposomes possess a pH-sensitive fluorophore
that allows the quantification of the conjugated liposomes
uptake within the endosomal compartment [107].
10. Antibody-Conjugated Nanoparticles in

Diagnosis
10.1. Cell Sorting. Without doubt cell sorting and bioseparation are the main applications for the antibody-conjugated
nanoparticles. As can be seen in Table 1, several companies
oer equipment for cell bioseparation.
In 1981, Kandzia et al. [108] described the use of
a monoclonal HLA-BW6 antibody coupled to albumincoated magnetite microspheres via surface-incorporated
Staphylococcus aureus Protein-A. The mixture of HLABW6 and -BW4 human peripheral blood lymphocytes was
incubated with these immunomicropheres and applied to a
glass column located in a magnetic field. Only HLA-BW4
lymphocytes passed through the column and were collected.
The recovered cells were 97% viable. Protein A (protein
isolated from the cell wall of Staphyloccocus aureus) was
chosen, because it interacts specifically with the Fc fragment
of the antibody.
However, in vivo experiments provided evidence of
competitive displacement of the same adsorbed antibody by
serum proteins, and the nanoparticles were cleared up to the
liver and spleen [109].
Coupling of a murine monoclonal antibody, HEA125,
specific for human epithelial cells, to magnetic beads (from
50 nm to a few microns) permits the positive selection of a
population containing essentially only tumour cells [110].
The problem with all cell types is that surface markers
are represented on most, if not all, of the cells. Therefore,
antibody-based magnetic separation is a powerful technique
for obtaining pure cell populations, but not all the cells of the
cell population [111].
10.2. Imaging. The diagnosis of cancer at early stages of
growth is a critical factor for obtaining optimal results
in therapy and for improving the chances of survival.
There are several imaging techniques that help physicians
in their diagnosis, including magnetic resonance imaging
(MRI), positron emission tomography (PET), computed
tomography (CT), ultrasound, radiography, photoacoustic
imaging, fluoroscopy, and so forth. In some of these
techniques antibody-conjugated nanoparticles may oer
increased selectivity and sensitivity.
10.2.1. Radio-Conjugated Antibody-Functionalized Nanoparticles. As early as 1981, Mach et al. [112] published the
first clinical results with 125I labelled anti-CEA monoclonal
antibodies, describing the potential applications of those
carriers. Nowadays, these vectors have been combined with
nanoparticles, thereby, providing new enhanced properties.
The work of Natarajan et al. [113] describes the
conjugation of commercial PEG-coated dextran-magnetic
nanoparticles with isotopically labelled recombinantly generated antibody fragments, di-scFv-c, for the imaging and
therapy (by applying magnetic hyperthermia) of anti-MUC1-expressing tumours. A chelator (DOTA) is linked to
the antibody fragment in the first step. Subsequently, the
conjugated antibody is incubated with a 111 In precursor
(isotopically labelled indium chloride) hybrid nanoparticle,
and finally, this radiolabelled antibody fragment is linked to
the previously described maleimide-functionalized magnetic
nanoparticle, by a covalent attachment to a free cysteine
of the fragment. The radiochelator is used to trace the
nanoparticles in vivo.
10.2.2. Targeted Contrast Agents for MRI. Among clinical
devices used for cancer diagnosis, Magnetic Resonance Imaging (MRI) stands out as a powerful tool, often superior to
other technologies. MRI is a noninvasive and nondestructive
powerful imaging modality, which provides internal images
of living organisms with no limits in the depth of analysis,
and with a resolution of 10100 m. It is versatile, as a wide
range of MRI modalities can be accessed, and 2D and 3D
imaging can be carried out [114].
MRI sensitivity can be improved by the use of contrast
agents (approximately 30% of all the clinical MR are assisted
by their use), which are active substances loaded with metal
ions usually as nanoparticulates. The inclusion of magnetic
nanoparticles as contrast agents for MRI prompted the
design of new targeted contrast agents. In 1998, monoclonal antibodies directed against epidermal growth factor
receptors (EGFRs), which are overexpressed in oesophageal
squamous cell carcinoma, were covalently linked to superparamagnetic nanoparticles, showing enhanced contrast for
tumour-specific MRI in a rat model bearing oesophageal
squamous cell carcinomas [115].
Isolated rat islets have been cultivated with immunomagnetic beads coated with antibody against rat MHC class I
antigen. Labelled islets were transplanted into the livers of
syngeneic rats providing islet images in vivo [116].
The detection of fibrin in vitro by MR in unstable
atherosclerotic plaques is possible by using paramagnetic
nanoparticles conjugated to an antifibrin antibody (1H10)
[117].
However, as mentioned above, the required dose of
the labelled antibody is still too high to make commercial
development realistic. Although biotechnology has advanced
tremendously, the low expression of antigens or receptors
and the limited sensitivity of the currently available relaxation enhancers are just some of the factors that call into
question the potential of antibodies or their fragments for
clinical MRI [118].
10.3. Sensing. Highly sensitive detection and accurate analysis of biomarker molecules in human fluid samples are
essential for the early detection, treatment, and management
of diseases [119].
In a biosensor, a ligand and a receptor bind together
in a reaction that is collected as a signal to a transductor using dierent methods, including optical, magnetic,
electrochemical, radioactive, piezoelectric, mechanical, mass
spectrometric, and so forth. Like any sensor, a biosensor
should be cheap, compact, selective, sensitive, portable,
reusable, and have a fast readout.
The introduction of nanoparticles in the molecular
diagnosis field has represented an advantage in many cases to
well-established detection techniques based on fluorophores,
such as Polymerase Chain Reaction (PCR) and EnzymeLinked ImmunoSorbent Assay (ELISA). Nanoparticles oer
their physical properties to the biosensor. In some cases,
nanoparticles are used simply as carriers of antibodies to
recognize them by association in biosensors. For example, in
1996, surface plasmon resonance (SPR) was used, through
the BIACORE biosensor, to demonstrate the specific
interaction between an anti-CD4 monoclonal antibody
(IOT4a), adsorbed on poly(methylidene malonate 2.1.2)
nanoparticles, and the CD4 molecule [120]. As can be seen
in Table 1, several companies oer equipment based on antibody conjugated nanoparticles for biosensing applications.
10.3.1. Optical Sensors. The enhancement of optical
immunosensors with nanoparticles was postulated as long
ago as 1997 by Kubitschko et al. [121] Subsequently, many
applications have demonstrated the superior capabilities of
nanoparticles.
Noble metal nanoparticles exhibit a strong UV-visible
absorption band that is not present in their bulk counterparts. For those nanoparticles localized, surface plasmon resonance excitation results in wavelength selective
absorption; large molar extinction coecients (superior to
fluorophores); and enhanced local electromagnetic fields on
the surface, responsible for intense signals in spectroscopy
[122].
Magnetic nanoparticles labelled with the near-infrared
fluorochrome Cy5.5 have been covalently bonded to anti
15
VCAM-1 antibodies by using EDC/Sulfo-NHS chemistry. An
eective delivery and labelling of activated endothelium was
demonstrated in vivo using a murine inflammatory model
[123]. The fluorescent probe was used to provide fluorescence confocal images of the m-TNF activated endothelium
in an ear model, and the magnetic part of the nanoparticles
was used to provide magnetic resonance (MR) contrast for
deep tissue imaging.
Gold nanoparticles are widely used in optical sensing,
due to their large light absorption and scattering crosssection in the surface plasmon resonance wavelength region.
The magnitude of light scattering by gold nanoparticles
can be orders of magnitude higher than light emission from
strongly fluorescing dyes [119]. They do not photobleach and
can be easily detected in as low as 1016 M concentration
[124].
In addition, gold nanoparticles have been used as
contrast agents for biomedical imaging using scanning
optical microscopy [125], multiphoton plasmon-resonance
microscopy [126], optical coherence microscopy and thirdharmonic microscopy [127]. Hirsch et al. [128] described
the use of antibodies conjugated to gold nanoparticles to
detect proteins in saline, serum, and whole blood by using
near infrared spectroscopy with a sensibility of up to subnanograms/mL.
Gold-coated silver nanoparticles, covalently attached to
goat anti-mouse IgG antibodies and to a reporter, were used
in biosensing applications by employing surface enhanced
Raman spectroscopy (SER) [129]. A capture antibody was
immobilized on a substrate and a positive detection was
observed when the goat IgG was present in the media.
Fluorescent reporter proteins embedded in polyacrylamide nanoparticles have been used for the sensing of
inorganic phosphate, by using the fluorescence resonance
energy transfer (FRET). The determination of inorganic
phosphate is necessary to elucidate metabolic processes
[130].
Anti-HER2 and anti-IgG gold-coated silica nanoparticle
conjugates have been used to measure the number of targeted
plasmonic nanoparticles that bind per cell, due to their
strong light scattering properties. The ability to quantify
this is crucial for improved diagnostic and therapeutic
results. The polarized angular-dependent light scattering
of the nanoparticles was studied using an automated
goniometer, because of the linear relationship between
the light scattering and the nanoparticle concentration
[131].
Anti-prostate specific antibodies have been conjugated
to gold nanoparticles and used to quantify the amount
of prostate specific antigen (an FDA-approved biomarker
for prostate cancer diagnosis) by utilizing dynamic light
scattering. Even with a small level of nanoparticle instability
and aggregation, a quantitative immunoassay is possible in
a protein matrix solution, by using dynamic light scattering
[119].
The scattering images and the absorption spectra
recorded from anti-EGFR conjugated gold nanoparticles,
incubated with cancerous (HOC 313 clone 8 and HSC 3 oral
epithelial cell lines) and noncancerous cells (nonmalignant
16
epithelial cell line HaCaT), are very dierent, and oer
potential techniques for cancer diagnosis [132].
The localized surface plasmon resonance absorbance
in the visible region has been used as an optical method
for the enhanced detection on an immunochromatographic
test strip, by using monoclonal anti-human gonadotropin
hormone (HCG) antibodies and monoclonal anti-human
total prostate specific antigen antibodies (anti-TPSA), conjugated to gold nanoparticles. The conjugation took place
by physical adsorption. The results showed a detection
limit of HCG at 1-0.1 ng/mL with the naked eye, and
a limit of 0.001 ng/mL (1 pg/mL) with the concentration
phenomena of the gold nanoparticles, using surface plasmon resonance measurements. TPSA was measured with
a limit of detection of 0.2 ng/mL [133]. The pregnancy
hormone was also determined, using magnetic nanoparticles
conjugated to anti-HCG antibodies with covalent coupling
[134].
Monoclonal mouse glycosylated antibodies have been
conjugated through the Fc region to gold nanoparticles
[135] and employed to produce nanoparticle labelling, using
dark field transmittance imaging. Those contrast agents were
validated in live NIH3T3 fibroblasts.
10.3.2. Mass Sensors. The most widely used mass biosensors
are quartz crystal microbalances (QCMs) and cantilevers. In
the former, a variation in the frequency of the resonance
is directly correlated to the mass deposited on its surface
(Sauerbrey equation). The QCM system is based on the
principle that the resonant frequency shifts of piezoelectric
crystal are directly proportional to the adsorbed molecular
mass [136].
For liquid detection, a thin film of the analyte on the
surface of the QCM is necessary for its accurate quantification. This adsorbed film may consist of a considerably
large amount of water, which is sensed as a mass uptake
by all QCMs. The measuring of several frequencies and
the dissipation (the dissipation, or damping, is the sum
of all energy losses in the system per oscillation cycle)
makes it possible to determine whether the adsorbed film
is rigid or water-rich (soft), which is not possible by
only looking at the frequency response. Therefore, the
evaluation of the dissipation is necessary in mass-based
biosensing applications, and it is possible to evaluate the
variation of the resonance frequency, after the deposition of
a moiety, and/or also the deflection of the cantilever after the
deposition. The damping problem also aects the cantilevers,
but currently commercial applications include a corrector
(named feedback) to subtract that contribution.
Piezoelectric QCMs have been used for direct detection
of anti-Toxoplasma gondii immunoglobulins. For patients
with weakened immune systems (including those with AIDS,
cancer, or other chronic illnesses), toxoplasmosis can be fatal.
Gold nanoparticles functionalized with Toxoplasma gondii
antibodies were incubated with infected rabbit serum and
blood. The nanoparticles are used to enhance the sensibility
of the mass sensor, based on the specific agglutination of
the antigen-coated gold nanoparticles on the QCM surface
[137].
The same transductors have been used for the piezoelectric immunoassay of carbohydrate antigen CA19-9 (an
important carbohydrate tumour marker expressed in several
kinds of carcinomas), by utilizing hybrid nanoparticles composed of poly-L-lysine/hydroxiapatite and carbon nanotubes
conjugated to mouse anti-CA19-9 antibodies [138]. The
nanocomposite acts as a scaold for protein immobilization.
QCMs have been used for the selective detection in serum
of the marker protein S100, which has been proposed
to help distinguish between ischemic and hemorrhagic
stroke [139]. A primary antibody was covalently attached
to the treated surface of the QCM and, in the presence
of the antigen, a streptavidin-coated magnetic nanoparticle
functionalizated with a secondary antibody was sandwiched,
enhancing the signal. The marker was measured in a
concentration as low as 0.2 ng/mL.
The surface of a QCM has been modified with a
layer of gold nanoparticles and protein A to achieve an
oriented attachment of antibodies (anti-CD3-FITC, antiCD5-FITC, anti-CD7-FITC, and mouse anti-human IgG1FITC) that recognize dierentiated antigens of phenotypic
lineages, or subsets of acute leukaemia [140]. The use of the
nanoparticles helps to obtain a correct orientation of the
antibodies and also in their regeneration.
The incorporation of colloidal gold nanoparticles into
hybrid matrices can retain the activity of biomolecules,
and enhance the immobilized amount of biomolecules. For
instance, Tang et al. [141] have described the use of gold
nanoparticles to enhance the signal for the recognition of
carcinoembryonic antigens, employing QCMs because of
the better immobilization of the antibodies on the surface
of the mass sensor. According to the authors, the use of
nanoparticles may enhance the immobilization density of
bound antibodies and, also, might serve as an intervening
spacer matrix to extend the immobilized antibody away
from the substrate matrix in the mobile phase, resulting in
binding sites more accessible to antigens.
Furthermore, the enhancement of the immobilization
phenomena by using nanoparticles in biosensing with QCMs
has been reported by other authors [142].
10.3.3. Electrochemical Sensors. Electrochemical sensors are
widely used for the detection of several biological molecules
of clinical interest, by specific recognition through oligonucleotide probes or antibodies immobilized on the detection
platform. The captured target is then recognised, in a
sandwich format, through its reaction with a secondary
antibody or DNA probe connected with easily detectable
electroactive nanoparticles. The detection mechanism is
based on the electrochemical stripping [143] (usually anodic
stripping voltammetry) of the heavy metal based nanoparticles. Depending on the transduction mechanism, the electrochemical sensors can use a potentiometric, amperometric, or
conductometric detection.
The antigen-antibody conjugation has been determined
by using a biomimetic surface (based on red blood cells)
sandwiched between gold nanoparticles deposited on a
carbon electrode surface and gold nanoparticles present
on the cell surface. These gold nanoparticles have been
functionalized with mammary cancer 15-3 antibody (antiCA15-3). In the presence of the antigen, the amperometric
response decreased with the augmentation of CA15-3 sample
concentration [144].
The work of Li et al. [145] describes the use of an electrochemical immunosensor for determining the macrophage
migration inhibitory factor (MIF), in the serum of patients
with rheumatoid arthritis. The surface of the gold electrode
of the sensor was coated with compounds of gold and
TiO2 nanoparticles and thionine (NGP-NTiP-Thi), followed
by adsorption of anti-MIF antibodies with IgM or IgG1
isotype. The measurements showed considerable levels of
sensitivity, selectivity, stability, and long-term maintenance
of bioactivity.
A reagentless immunosensor for rapid determination of
carbohydrate antigen 19-9 (CA19-9) in human serum was
described by Du et al. [146]. This strategy was based on the
immobilization of antibody in colloidal gold nanoparticlemodified carbon paste electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was conjugated
to a CA19-9 antibody. The nanoparticles were ecient in
preserving the activity of immobilized biomolecules.
A separation-free electrochemical immunosensor for
carcinoma antigen-125 (CA125) was proposed based on
the immobilization of the CA125 antigen, on colloidal
gold nanoparticles, which had been stabilized with cellulose acetate membrane on a glassy carbon electrode. The
immunosensor showed good accuracy, acceptable sensitivity,
precision, reproducibility, and storage stability [147].
10.3.4. Magnetic Sensors. Kotitz et al. [148] proposed a magnetic relaxation/remanence immunoassay, with a superconducting quantum interference device (SQUID) as magnetic
sensor. In this technique, an immobilized target is immersed
in a suspension of superparamagnetic nanoparticles bound
to antibodies specific to that target. The SQUID detects the
magnetic field from the particles bound to the target. An
improvement of this technique was achieved by Chemla et al.
[149] employing a high-transition temperature SQUID for
the ultrasensitive detection of FLAG . The antibody recognizes the FLAG epitope located on FLAG -tagged fusion
proteins. The disadvantage of this detection technique is that
it does not allow the simultaneous detection of multiple
analytes. Subsequently, Kim and Park [150] described the
use of a microfluidic system for the simultaneous detection
of multiple antibodies (goat polyclonal anti-mouse IgG
(H+L) F(ab )2 fragments and goat polyclonal anti-rabbit
IgG (H+L) F(ab )2 ) attached to magnetic and non-magnetic
(fluorescent) nanoparticles by measuring the magnetic
force.
The use of magnetoresistance for the biodetection of the
antigen-antibody coupling was postulated in 1998 by Baselt
et al. [151].
Perez et al. [152], after measuring magnetic relaxation,
were able to describe the sensing of magnetic nanoparticles
conjugated with anti-adenovirus 5 (ADV-5) or anti-herpes
simplex virus 1 (HSV-1) antibodies that, after recognition,
agglomerate in the presence of the antigen with, as a consequence, an increase in the relaxation time. The technique
17
could be of potential use for detecting the distribution of
HSV and ADV viruses by MRI in vivo.
Multiplexed protein assays are detectable with the commercial equipment, marketed as the Verigene Reader
(Nanosphere Inc., Northbrook, Ill, USA). The protein of
interest present in serum is simultaneously attached via
hybridization to: magnetic nanoparticles surface functionalized with primary antibodies that recognize the protein
of interest; gold nanoparticles functionalized with both
secondary antibodies that recognize the protein of interest;
and to oliglonucleotides that have dierent base sequences
to identify each protein target in the sample. Once the
nanoparticles have sandwiched a protein target, a permanent
magnet is used to separate and isolate it from the rest.
The optical detection is based on the capture of the scattered light from the gold-based nanoparticles (scanometric
method) [153].
Magnetic nanoparticles coated with DMSA (meso-2,3dimercaptosuccinic acid) and covalently bonded to antihuman cardiac troponin-I have been synthesized, and their
biological activity has been evaluated by ELISA and Western
Blotting. The stability studies revealed that after 300 days
the antibodies on the magnetic nanoparticles retained their
biological activity [154].
11. Antibody Conjugation to Nanoparticles

Bioconjugation can take place by means of adsorption
(at the isoelectrical point of the antibody via electrostatic
interaction), by direct covalent linkage between the surface
of the nanoparticle and the antibody, or by using adapter
molecules. The use of adapter molecules generally involves
streptavidin and biotin for the formation of the complex.
Biotinylated antibodies are commercially provided. As an
example, biotin-labelled polyclonal goat anti-Escherichia coli
antibodies have been attached to streptavidin-coated magnetic nanoparticles, and used for the separation and selective
quantification of Escherichia coli O157:H7 in ground beef in
the presence of other bacteria [155].
Moreover, gelatine and human serum albumin nanoparticles have been surface modified by the covalent attachment
TM
enabling the
of the biotin-binding protein NeutrAvidin
binding of biotinylated anti-HER2 antibodies, showing
intracellular specific cell targeting [156]. This group also
described the selective targeting of the same nanoparticles
conjugated to antibodies, specific for the CD3 antigen on
CD3-positive human T-cell leukaemia cells and primary
T-lymphocytes [157]. The same biotin-binding protein
has been attached to the surface of poly(DL-lactic acid)
nanoparticles thanks to the presence of thiol functions on
their surface [158].
The clear advantage of using covalent linkages compared
to physical adsorption is that the linkage prevents the
competitive displacement of the adsorbed antibodies by
blood components, which occurs for adsorbed antibodies.
Optimal bioconjugation would involve the covalent
attachment of the antibody through the Fc (fragment:
crystallizable) region, leaving the antigen-binding site Fab
(fragment: antigen binding) region oriented to the medium
18
to preserve its full function, and in a ratio of one to
one to allow a direct quantification. In addition, the
use of a spacer is highly recommended to enhance the
probabilities of the antibody finding its corresponding
antigen. Hence, oriented covalent binding may increase
antibody stability and control the available protein binding
sites.
The excellent review of Nobs et al. [159] describes the
current methods for attaching targeting ligands to liposomes
and nanoparticles.
11.1. Random Antibody Orientation. It is possible to prepare
antibody-conjugated nanoparticles, where the antibodies are
randomly attached to the surface of the nanoparticle, by
using numerous approaches, however the following are the
most common.
(1) Formation of a Schi s base between the primary
amine of the antibody and the free aldehyde groups of
the glutaraldehyde (bifunctional crosslinker) present on the
surface of the amino-functionalized nanoparticle [160].
(2) Formation of an amide bond between the carboxylated nanoparticles and the amino groups of the antibodies,
using EDAC or EDC (1-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride) as cross-linking agents [161].
Following this procedure, commercial carboxylated magnetic nanoparticles have been covalently attached to polyclonal antibodies used against a sulphonamide-based antibiotic, which can be present in dairy products, for the targeted
capture and enrichment of those antimicrobials in milk
and hair samples [162]. Invasive epithelial breast tumour
cells have also been targeted with polylactic-co-glycolic acid
(PLGA) conjugated to a fluorochrome-labelled monoclonal
antibody, which recognizes cytokeratins expressed in breast
epithelial cell lines and breast tumour cells. Physical adsorption and covalent linkage, with EDC as a linking reagent to
form an amide bond, were used for comparison. A binding
assay using cell lysate revealed that the recognition properties
were preserved only for nanoparticles with the monoclonal
antibody adsorbed on their surface [163], corroborating
that the correct orientation of the antibody is necessary.
Carbodiimide chemistry was used to attach polyclonal antiantibodies to the siglec-7 (CD33-like), and to dye-loaded
PLGA nanoparticles, as well as to corroborate the cell
internalization pathway through endosomal compartments,
by confocal microscopy on mouse embryonic fibroblasts
[164].
(3) Formation of an isourea derivative between the
hydroxyl surface groups of the nanoparticles and the amino
groups of the antibodies, using cyanogen bromide [165].
(4) Poly(lactic acid) nanoparticles have been functionalizated with carboxyl groups and attached to a nucleophile
(cysteine or cystamine) to provide thiol groups via an
amide bond [166]. Anti-HER2 (Herceptin ) and antiCD20 (Mabthera ) monoclonal antibodies were covalently
coupled to two kinds of labelled poly(DL-lactic acid)
nanoparticles. Thiol groups were introduced on the surface
of the nanoparticles by a two-step carbodiimide reaction.
The covalent linkage was achieved using a bifunctional crosslinker. In vitro results showed a dierent cellular localization
for both nanoparticles depending on the type of antigenantibody interaction involved [167].
The work of Fuentes et al. [168] describes the biological
activity of IgG anti-horseradish peroxidase attached to
activated supports, using the oriented and random coupling
chemistry of the antibodies, opening up the spectrum of
alternatives for the attachment.
11.2. Oriented Conjugation of the Antibody. It is an advantage
if the attachment of the antibody through its Fc region to the
nanoparticle is fairly easy, without modifying the antibody
activity, stable in a media with high ionic concentration, and
allowing a high load of antibody per nanoparticle.
In addition to the use of protein A, which interacts specifically with the Fc fragment of the antibody as mentioned
above, the literature describes dierent strategies for the
directional attachment of the antibodies with the antigenbinding sites on the Fab portion directed outward from
the surface of the nanoparticle and, therefore, available for
targeting. These strategies include the surface functionalization of the nanoparticles to render aldehydes, epoxides,
thiols, and so forth. It is possible to achieve an oriented
conjugation with those functional groups using the following
bonds: aldehydes to the lysine-rich region of the antibody;
epoxides to the histidine-rich region of the antibody; and
thiols to the centre thiol groups of the antibody obtained by
reduction. Monoclonal mouse glycosylated antibodies have
been conjugated through the Fc region to gold nanoparticles,
employing a covalent attachment of a heterofunctional
linker (an amide-bonded adipic hydrazide), and an alkane
terminating in a dithiol tether via gold-thiol bonds [135].
Hetero-functional epoxy-chelate and immobilization by the
sugar chain on amino groups has been demonstrated as
another strategy to achieve oriented coupling of antibodies
[168].
The other alternative, where a specific orientation of
the antibody is not necessary, is the direct attachment
of the antibodys fragments (Fabs) to the surface of the
nanoparticles.
For this reason, long-circulating (Stealth ) immunoliposomes were targeted against the B-cell antigen CD19, via
a whole HD37 monoclonal antibody (HD37 mAb), versus
its Fab fragment (HD37 Fab ) or an HD37c-mycCys
His5 single-chain Fv fragment (scFv, HD37-CCH) directed
against the same epitope [169]. The coupling through the
Fab fragment showed the longest circulation half-life of the
conjugated nanoparticles tested, and appeared to be slightly
more eective than the others in prolonging survival times.
12. Conclusions
Monoclonal antibodies (mAbs), or molecules derived from
them, are basic elements for laboratories of clinical diagnosis
and research, but are also some of the drugs with the greatest
impact in the treatment of dierent human diseases, including cancer. Currently, there are at least 26 mAbs approved for
human therapy, the majority of which are recombinant or
humanized antibodies. These antibodies have mainly human
sequences in their structure, and a minority of murine
ones, in order to eliminate or reduce immune response.
Yet, the antibodies have a massive therapeutic potential and,
currently, 150 are already in dierent phases of clinical trials
and it is likely that, in the near future, many of them will
be approved by the regulatory authorities for therapeutic
use against dierent diseases. Currently, nanotechnology is
undergoing rapid an enormous development, making a wide
variety of applications possible, especially in the field of in
vitro and in vivo diagnosis and even in human therapy. The
twentieth century was characterized by the vast expansion
of the production of mAbs for diagnostic and research
techniques; from the final years of that century and in the
twenty-first century we have entered the era of mAbs and
nanoparticles in human therapy. In this review, we have
tried to summarize the most commonly used techniques for
the production of these drugs, to show the ones with more
advantages, as well as to provide an update of the monoclonal
antibodies currently approved for therapeutic use.
19
[10]
[11]
[12]
[13]
[14]
[15]
[16]
Acknowledgments
The authors would like to thank the Spanish Nanoscience
Action NAN200409270-C3-1/2, the Xunta de Galicia
(PGIDIT06TMT31402PR), and the Consolider Ingenio
2010 Program (CSD2006-00012) for supporting this study.
M. Arruebo would like to acknowledge the support of the
y Cajal Program (order ECI/158/2005). The
2006 Ramon
authors Manuel Arruebo and Monica

Valladares contributed
equally to the manuscript.
[17]
[18]
[19]
[20]
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Antibody-Conjugated Nanoparticles For Biomedical Applications

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Hindawi Publishing Corporation

Manuel Arruebo,1 Monica

Correspondence should be addressed to Manuel Arruebo, arruebom@unizar.es

nanoparticles show larger surface area to volume ratio, which

2. Antibodies: Structure and Function

Figure 1: Structure of an Ig molecule carrying two heavy and two

the constant domain (CL ). The heavy chain (H) contains a

Figure 2: Schematic description of the synthesis of monoclonal

workshop. As examples, a helper T lymphocyte is known

4. Monoclonal Antibodies in Therapy

configuration is altered, but humanized antibodies have the

Figure 3: Genetic construction of dierent antibody fragments.

origin so that the resulting molecule is less strange for

Primatized Antibodies. Primatized antibodies are the result

5. Human Monoclonal Antibodies

6. Therapeutic Uses of Monoclonal Antibodies

7. Monoclonal Antobodies Approved for

8. Nanoparticles and Antibodies

Chimeric 7E3 directed against platelet glycoprotein IIb/IIIa

Mouse IgG2a anti-CD3. Transplantation Rejection

Mouse Fab-Indio111 , directed against human heart miosin

Mouse Ig Fragment-Tecnecio99 . Directed against

Mouse Ab-Indio111 . Detection of prostate tumour

Humanized IgG1 anti-IL-2. To avoid transplant rejection

Chimeric Ig antiCD20 for Non-Hodgkin lymphomas

Chimeric anti-IL-2(CD25). To avoid renal transplant

Humanized IgG1 ; respiratory syncitial virus

Humanized IgG anti-HER2; breast cancer

Centocor (Johnson &

Chimeric anti TNF-. Rheumatoid arthritis, Crohns disease

Humanized Ig anti CD33; acute myeloid leukaemia

Humanized Ig anti CD52; chronic lymphocitic leukaemia

Human anti TNF-. Rheumatoid arthritis

Mouse Ig-Itrio 90 anti CD20. Non-Hodgkin lymphoma

Murine Ig-Iodo131 anti CD20. Non-Hodgkin lymphoma

IgG1 directed against EGFR; colorectal tumour

Humanized anti VEG; tumours

Humanized anti CD49d. Multiple Sclerosis, Chrons disease

Humanized anti VEGF-A. Wet Macular Degeneration

Human anti EGFR (epidermal growth factor receptor).

Bangs Laboratories Inc.

the selective accumulation of nanoparticles of sizes generally

Can find specific target

Figure 5: Advantages in the conjugation of nanoparticles to antibodies.

the number of antigens recognised by tumour-associated

Biochemistry & molecular biology

nanoparticle, one of them, monoclonal anti-TfR, to

10. Antibody-Conjugated Nanoparticles in

11. Antibody Conjugation to Nanoparticles

authors Manuel Arruebo and Monica

gondii antibodies using gold nanoparticles, Biosensors and

[167] A. Cirstoiu-Hapca, L. Bossy-Nobs, F. Buchegger, R. Gurny,

Hindawi Publishing Corporation

Hindawi Publishing Corporation

Hindawi Publishing Corporation

Submit your manuscripts at

Materials Science and Engineering

Hindawi Publishing Corporation

Hindawi Publishing Corporation

Hindawi Publishing Corporation

Hindawi Publishing Corporation

Hindawi Publishing Corporation

Hindawi Publishing Corporation